Polypeptides obtained from eukaryotic cells are produced as glycosylated polypeptides. The glycostructures are attached to the amino acid backbone as post-translational enzymatic modification.
The glycosyltransferases are recognized as a functional family of estimated 250-300 different intracellular, membrane-bound enzymes that participate in the coordinate biosynthesis of the glycostructures of polypeptides, including glycoproteins, proteoglycans and glycolipids. The glycosyltransferases are classified into groups based on their nucleotide monosaccharide donor specificity. For example, the galactosyltransferases are the subset of glycosyltransferases that use UDP-galactose as the activated monosaccharide donor whereas the sialyltransferases use CMP-sialic acid and the fucosyltransferases use GDP-fucose (Shaper, N. L., et al., J. Mamm. Gland Biol. Neopl. 3 (1998) 315-324).
The modification of alpha-galactosyl epitopes on various mammalian cells is of particular interest, since as much as 1% of circulating IgG antibodies in humans interact with this oligosaccharide residue. This natural antibody, designated “anti-Gal”, was previously found to bind to terminal Gal(α1,3)Gal(β1,4)GlcNAc-R on biochemically defined glycolipids (Galili, U., et al., J. Exp. Med. 162 (1985) 573-582; Galili, U., et al., J. Exp. Med. 165 (1987) 693-704). Measurement of the binding of radiolabeled Bandeiraea (Griffonia) simplicifolia IB4 lectin to the various nucleated cells suggests that cells binding anti-Gal express 106 to 3.5×107 alpha-galactosyl epitopes, most of which, based on the anti-Gal specificity, seem to have the structure of Gal(α1,3)Gal(β1,4)GlcNAc-R. The absence of these epitopes from human cells results from diminished activity of the enzyme (α1,3)galactosyltransferase (Galili, U., et al., J. Biol. Chem. 263 (1988) 17755-17762).
The synthesis of the Gal(α1,3)epitope in the Golgi apparatus of cells of murine origin (Cummings, R. D. and Mattox, S. A., J. Biol. Chem. 263 (1988) 511-519; Blake, D. A., and Goldstein, I. J., J. Biol. Chem. 256 (1981) 5387-5393; Elices, M. J., Blake, D. A., and Goldstein, I. J. J. Biol. Chem. 261 (1986) 6064-6072), leporine origin (Basu, M., and Basu, S., J. Biol. Chem. 248 (1973) 1700-1706; Betteridge, A., and Watkins, W. M., Eur. J. Biochem. 132 (1983) 29-35), porcine origin and bovine origin (Blanken, W. M., and Van den Eijnden, D. H., J. Biol. Chem. 260 (1985) 12927-12934) has been demonstrated to be catalyzed by the enzyme (α1,3)galactosyltransferase.
In polypeptides which are intended for application to humans the presence of (α1,3)glycosidically bound terminal galactose residues should be minimized as this glycostructure will elicit a response by the human immune system. This can be achieved, for example, by the time-consuming development of cell lines for the recombinant production of the therapeutic polypeptide which do not introduce (α1,3)glycosidically bound terminal galactose residues in the glycostructures of the therapeutic polypeptide. With chromatographic method generally used in the downstream processing of the crude polypeptide the Gal(α1,3)-containing glycostructures cannot be removed.
In EP 0 255 153 a process for producing α-galactosidase capable of decreasing the galactose content of galactomannans by splitting off 1,6 linked alpha-D-galactopyranosyl units attached to a main chain of 1,4 linked beta-D-mannopyranosyl units is reported. A method for clinical examination based on the structures of immunoglobulin G-linked oligosaccharides is reported in EP 0 698 793. In EP 1 878 747 glyco-engineered antibodies are reported. Selective marking of immunoglobulin glycans is reported in WO 2007/071347. In WO 1997/016064 methods and compositions for the reduction of xenotransplantation rejection are reported. Antibody preparations with substantially homogeneous and unsialylated glycoforms, such as G0 and G2, which are prepared by enzymatic treatment, expression under certain conditions, use of particular host cells, and contact with serum, are reported in WO 2007/024743.
In WO 2008/057634 polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods are reported. Proteolysis resistant antibody preparations are reported in WO 2007/024743.